Half-life extension using serum albumin-binding DARPin® domains

Abstract

A long systemic half-life is key for therapeutic proteins. To that end we have generated serum albumin-binding designed ankyrin repeat domains. These domains bind serum albumin of different species with nanomolar affinities, and have significantly improved pharmacokinetic properties both in mouse and cynomolgus monkey compared to non-serum albumin-binding DARPin® domains. In addition, they exhibit high thermal stability and long storage stability, which is an essential feature for their use in drug development. Covalently linking a serum albumin-binding DARPin® domain to domains with other target specificities results in improvements of multiple orders of magnitude in exposure and terminal half-life, both in mouse and cynomolgus monkey. Pharmacokinetic assessment of such constructs revealed terminal half-life values ranging from 27 h to 80 h in mouse, and from 2.6 days to 20 days in cynomolgus monkey. Extrapolation by allometric scaling on these findings suggests terminal half-life values of 5-50 days in human, indicating that pharmacokinetic properties in the range of monoclonal antibodies can be achieved with DARPin® drug candidates. Such serum albumin-binding DARPin® domains are thus valuable tools for the generation of multi-functional drugs with an extended in vivo half-life.

Animal models for evaluation of albumin-based therapeutics

“…2d). Examples of such molecules are albumin-binding domain [42,43], albumin-binding lipids and peptides [6,44], albuminbinding designed ankyrin repeat proteins (DARPin) [45] and different forms of antibodyderived fragments with specificity for albumin [46][47][48][49]. To obtain extended half-life using this strategy, it is important that binding to albumin does not interfere with the FcRnalbumin interaction and binding needs to be retained at neutral pH as well as at lower pH encountered in the endosomes.…”

“…Albumin-binding molecules and attached drugs may be evaluated in WT mice if they bind to MSA. For instance, ribosome display was used to select albumin-binding DARPin molecules that bound albumin of different species with nM affinities at both pH 6.0 and pH 7.4 [45]. When an albumin-binding DARPin was fused to a DARPin with no specificity, the fusion exhibited a half-life of 44 hours in WT mice, which is similar to that reported for MSA (Table 1), while a non-albumin binding DARPin was eliminated in minutes [45].…”

Design and characterization of MP0250, a tri-specific anti-HGF/anti-VEGF DARPin® drug candidate

“…The background on serum albumin-binding DARPin Ò domains is described in detail elsewhere. 30 We applied the approach to MP0250, using two HSA-binding DARPin Ò domains flanking the other two domains. We analyzed the pharmacokinetic properties of MP0250 in single dose experiments in both mouse and cynomolgus monkey.…”

Serum albumin‐binding V H Hs with variable pH sensitivities enable tailored half‐life extension of biologics

“…Our data provide insights into the relationship between serum albumin binding affinity and serum half‐life extension: measurements of albumin‐binding affinity at acidic pH (5.5‐6.0) may be more predictive of half‐life than measurements at neutral pH. This may partially explain why previous studies have struggled to correlate serum albumin binding affinity with serum half‐life, with large variations in binding at neutral pH having only minor impacts on pharmacokinetic profiles 13,20,23 . In addition, our data clearly demonstrate that modest half‐life extension of biologics can be achieved through a novel mechanism involving interaction with albumin in serum but with no apparent contribution of endosomal recycling.…”

“…Many small serum albumin‐binding proteins (primarily antibody fragments) have been successfully used to extend the serum half‐lives of biologics, including streptococcal albumin‐binding domains (ABDs), 11,12 DARPins, 13 immunoglobulin variable heavy chain (V H ) and variable light chain (V L ) domains, 14,15 camelid heavy chain‐only variable (V H H) domains, 16 shark new antigen receptor variable (V NAR ) domains, 17 a fragment variable (Fv), 18 and a fragment antigen binding (Fab) 19,20 . Serum albumin‐binding proteins offer several important advantages over Fc fusions.…”